Proteinic drug delivery system using membrane mimetics

ABSTRACT

A mixed liposome pharmaceutical formulation with multilamellar vesicles, comprises a proteinic pharmaceutical agent, water, an alkali metal lauryl sulphate in a concentration of from 1 to 10 wt./wt. %, at least one membrane-mimetic amphiphile and at least one phospholipid. The membrane-mimetic amphiphile is hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide, sodium cocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylene dihydroxypropyl stearammonium chloride, dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA, gamma-linoleic acid, borage oil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyl lactylates, alkaline earth metal isostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borage amidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid, trihydroxy-oxo-cholanylglycine and alkali metal salts thereof, and octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanol X-oleyl ether, wherein X is from 9 to 20, or combinations thereof. The phospholipid is phospolipid GLA, phosphatidyl serine, phosphatidylethanolamine, inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin, triolein, lecithin, saturated lecithin and lysolecithin, or a combination thereof. The amount of each membrane mimetic amphiphile and phospholipid is present 1 to 10 wt./wt. % of the total formulation, and the total concentration of membrane mimetic amphiphiles and phospholipids is less than 50 wt./wt. % of the formulation.

FIELD OF THE INVENTION

The present invention relates to an improved delivery system for theadministration of large-molecule pharmaceuticals, e.g. peptidic drugs,vaccines and hormones. In particular it relates to pharmaceuticals whichmay be administered through the oral and nasal membranes, or bypulmonary access.

BACKGROUND TO THE INVENTION

New methods of delivering large macromolecules (proteins and peptides)continue to be sought. One of the avenues investigated concerns the useof membrane-mimetic amphiphiles. A study of membrane-mimetic amphiphilesextends back to the first decade of the 20th century. Experiments usingphysical and chemical methods have shown that such molecules assumepreferred arrays in the presence of water. Formation of these arrays,which includes micelles, monolayers and bimolecular layers is driven bythe need of the polar head groups, which may be ionogenic or not, toassociate with water, and the need of the polar hydrophobic tails to beexcluded from water, (Small, D; Handbook of Lipid Research, vol. 4,1986; Tanford, J: The Hydrophobic Effect, John Wiley & Sons, 1980;Fendler, J. Membrane Chemistry, 1982). Exactly which type of structureis assumed depends on upon the nature of the amphiphile, itsconcentration, the presence of other amphiphiles, temperature and thepresence of salts and other solutes in the aqueous phase.

Membrane-mimetic amphiphiles include molecules that are insoluble inwater but can take up water, and molecules that have appreciablesolubility in water under limiting conditions. The former amphiphiles donot form molecularly disperse solutions in water but may swellconsiderably with water to form lamellar phases. The latter amphiphilescan, at some temperatures, form solutions of dispersed monomers in waterand often undergo the following sequence as the concentration in wateris increased: monomeric solution to micellar solution. The manufactureof non-phospholipid liposomes, depends on the manipulation ofenvironmental variables (e.g. temperature, hydration and composition) inan appropriate temporal sequence so as to cause non-phospholipidamphiphiles to form liposomal structures.

Gebicki et al. (Nature, 243, 232, 1973: Chem. Phys. Lipids, 16, 142,1976; Biochem. Biophys. Res. Commun. 80, 704, 1978; Biochemistry, 17,3759, 1978) demonstrated the formation of water containing vesiclesenclosed by oleic acid. Others, as disclosed for example in U.S. Pat.Nos. 4,772,471 and 4,830,857, and in J. Microencapsul. 4, 321, 1987,have made lipid vesicles from single tailed ether or esters derivativesof polyglycerol. These liposomes were found suitable for cosmeticproducts. Murakami et al (J. Am. Chem. Soc, 101, 4030, 1979; J. Am OilChem Soc. 66, 599, 1989) formed single compartment vesicles with one ormore bilayer walls composed of cationic amphiphiles involving amino acidresidues. Kaler et al (Science, 245, 1371, 1989) demonstrated thatappropriate aqueous mixtures of single-tailed cationic and anionicsurfactants spontaneously form single-walled vesicles, presumably viasalt formation. Others have developed methods for manufacture ofpaucilamellar, non-phospholipid liposomes that can be formed from avariety of amphiphiles as well as from certain phospholipids. Theliposomes have two or more membranes surrounding an amorphous core, eachmembrane being composed of amphiphile molecules in bilayer array. Thecore accounts for most of the vesicle volume and encapsulatingsubstances.

The above-mentioned non-phospholipid based liposomes are mainly used forthe delivery of moisturizers and cosmetic ingredients used topically orexternally as creams or moisturizers. In some cases such liposomes maybe used as an ointment for delivery of some pharmaceutical products.Many ingredients utilized in the above products have been found to beinadmissible in the human body and are not approved by the regulatoryagencies around the world for the purpose of oral administration and asa vehicle for delivery of macromolecules (proteins and peptides) as lifesaving therapeutics. Furthermore, other non-phospholipid based liposomeshave been developed for non-pharmaceutical applications, e.g.water-borne oil paints, surface cleansers, heavy duty industrialcleansers and skin-cleansing detergents.

Certain aspects of the present invention aims at the development of oralcompositions consisting of mixture of certain non-phospholipid basedmembrane-mimetic amphiphiles (suitable and approved by the regulatingagencies for oral formulation of human pharmaceutical products) incombination of specific phospholipids to form multilamellar liposomeswhich are very stable and are smaller than the pores of thegastrointestinal (GI) tract.

Relatively very little progress has been made in reaching the target ofsafe and effective oral formulations for peptides and proteins. Themajor barriers to developing oral formulations for proteins and peptidesinclude poor intrinsic permeability, lumenal and cellular enzymaticdegradation, rapid clearance, and chemical stability in the GI tract.Pharmaceutical approaches to address these barriers, which have beensuccessful with traditional small, organic drug molecules, have notreadily translated into effective peptide and protein formulations.Although the challenges are significant, the potential therapeuticbenefits remain high especially in the field of diabetes treatment usinginsulin.

Researchers have explored various administration routes other thaninjection for proteins and peptides. These routes include administrationthrough oral, intranasal, rectal, vaginal cavities for the effectivedelivery of large molecules. Out of the above four mentioned routes oraland nasal cavities have been of greatest interest. Both the oral andnasal membranes offer advantages over other routes of administration.For example, drugs administered through these membranes have a rapidonset of action, provide therapeutic plasma levels, avoid a first passeffect of hepatic metabolism, and avoid exposure of the drug to ahostile GI environment. Additional advantages include easy access to themembrane sites so that the drug can be applied, localized and removedeasily. Further, there is a good potential for prolonged delivery oflarge molecules through these membranes.

The oral routes have received far more attention than have the otherroutes. The sublingual mucosa includes the membrane of ventral surfaceof the tongue and the floor of the mouth whereas the buccal mucosaconstitutes the lining of the cheek. The sublingual mucosa is relativelypermeable thus giving rapid absorption and acceptable bioavailability ofmany drugs. Further, the sublingual mucosa is convenient, acceptable andeasily accessible. This route has been investigated clinically for thedelivery of a substantial number of drugs.

Various mechanisms of action of penetration of large molecules usingenhancers have been proposed. These mechanisms of action, at least forprotein and peptidic drugs include (1) reducing viscosity and/orelasticity of mucous layer, (2) facilitating transcellular transport byincreasing the fluidity of the lipid bilayer of membranes, (3)facilitating paracellular transport by altering tight junction acrossthe epithelial cell layer, (4) overcoming enzymatic barriers, and (5)increasing the thermodynamic activity of drugs (Critical Rev. 117-125,1992).

Many penetration enhancers have been tested so far and some have beenfound effective in facilitating mucosal administration of largemolecular drugs. However, hardly any penetration enhancing products havereached the market place. Reasons for this include lack of asatisfactory safety profile respecting irritation, lowering of thebarrier function, and impairment of the mucocilliary clearanceprotective mechanism. It has been found that some of the popularpenetration enhancers, especially those related to bile salts, and someprotein solubilizing agents, impart an extremely bitter and unpleasanttaste. This makes their use impossible for human consumption on a day today basis. Several approaches were utilized to improve the taste of thebile salts based delivery systems, but none of them are commerciallyacceptable for human consumption to date. Approaches utilized includepatches for buccal mucosa, bilayer tablets, controlled release tablets,liposome formulations, use of protease inhibitors, bucally administeredfilm patch devices, and various polymer matrices. Further the problem iscompounded because of the localized side effect of a patch which oftenresults in severe tissue damage in the mouth.

SUMMARY OF THE INVENTION

Accordingly the present invention provides a mixed liposomepharmaceutical formulation with multilamellar vesicles, comprising aproteinic pharmaceutical agent, water, an alkali metal lauryl sulphatein a concentration of from 1 to 10 wt./wt. % of the total formulation,at least one membrane-mimetic amphiphile and at least one phospholipid,

wherein the membrane-mimetic amphiphile is selected from the groupconsisting of hyaluronic acid, pharmaceutically acceptable salts ofhyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide,sodium cocoamphopropionate, bishydroxypropyl dihydroxypropylstearammonium chloride, polyoxyethylene dihydroxypropyl stearammoniumchloride, dioctadecyldimethylammonium chloride, sulphosuccinates,stearamide DEA, gamma-linoleic acid, borage oil, evening of primroseoil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkalimetal isostearyl lactylates, alkaline earth metal isostearyl lactylates,panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammoniumchloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borageamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropylphosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid,trihydroxy-oxo-cholanylglycine and alkali metal salts thereof,octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanolX-oleyl ether, wherein X is from 9 to 20, and cobinations thereof, and

wherein the phospholipid is selected from the group consisting of,phospholipid GLA (glycolic, lactic acid), phosphatidyl serine,phosphatidylethanolamine, inositolphosphatides,dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin,triolein, unsaturated lecithin, saturated lecithin and lysolecithin, andcombinations thereof, and

wherein the amount of each membrane-mimetic amphiphile and phospholipidis present in a concentration of from 1 to 10 wt./wt. % of the totalformulation, and the total concentration of membrane-mimetic amphiphilesand phospholipids is less than 50 wt./wt. % of the formulation.

Preferably the mixed liposome pharmaceutical formulation has a pH ofbetween 6.0 and 7.0.

The preferred number of membrane mimetic amphiphiles are from 2 to 5.

The preferred number of phospholipids are from 1 to 4.

In one embodiment, the alkali metal lauryl sulphate is sodium laurylsulphate.

In a preferred embodiment at least one protease inhibitor is added tothe formulation to inhibit degradation of the pharmaceutical agent bythe action of proteolytic enzymes. Of the known protease inhibitors,most are effective at concentrations of from 1 to 3 wt./wt. % of theformulation.

Non-limiting examples of effective protease inhibitors are bacitracin,soyabean trypsin, aprotinin and bacitracin derivatives, e.g. bacitracinmethylene disalicylate. Bacitracin is the most effective of those namedwhen used in concentrations of from 1.5 to 2 wt./wt. %. Soyabean trypsinand aprotinin may be used in concentrations of about 1 to 2 wt./wt. % ofthe formulation.

In one embodiment, the membrane-mimetic amphiphile is selected from thegroup consisting of hyaluronic acid, pharmaceutically acceptable saltsof hyaluronic acid and mixtures thereof, the concentration suchabsorption enhancing compound being from about 1 to about 5 wt./wt. %.

In another embodiment, suitable for delivery through oral mucosalmembranes, the formulation contains sodium lauryl sulphate, andcombinations selected from the group consisting of:

i) sodium salt of trihydroxy-oxo-cholanyl glycine, sphingomyelin andstearamide DEA;

ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA;

iii) ceramide and stearamidopropyl phosphatidyl PG-diammonium chloride;

iv) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin;

v) octylphenoxypolyethoxyethanol and saturated lecithin;

vi) sodium hyaluronate, polydecanol 9-lauryl ether, lecithin and eveningof primrose oil; and

vii) monoolein, saturated lecithin, sodium hyaluronate and evening ofprimrose oil.

In yet another embodiment, suitable for topical delivery transdermally,the formulation contains sodium lauryl sulphate and combinationsselected from the group consisting of:

i) lecithin, sodium hyaluronate, glycolic acid and propylene glycol; and

ii) sodium hyaluronate, sphingomyelin, glycolic acid and propyleneglycol.

Preferably the lecithin is saturated lecithin.

It will be recognized by those skilled in the art that for manypharmaceutical compositions it is usual to add at least one antioxidantto prevent degradation and oxidation of the pharmaceutically activeingredients. It will also be understood by those skilled in the art thatcolorants, flavouring agents and non-therapeutic amounts of othercompounds may be included in the formulation.

In one embodiment the antioxidant is selected from the group consistingof tocopherol, deteroxime mesylate, methyl paraben, ethyl paraben andascorbic acid and mixtures thereof. A preferred antioxidant istocopherol.

The formulation suitable for delivery through oral mucosal membranes maybe in chewable form, in which case it will be necessary to addingredients suitable for such form. Such ingredients include guar gum,powdered acacia, carrageenin, beeswax and xanthan gum.

The proteinic pharmaceutical agent may be selected from a wide varietyof macromolecular agents, depending on the disorder being treated,generally with molecular weights greater than about 1000 and especiallybetween about 1000 and 2 000 000. Pharmaceutical agents useful in thepresent invention include insulin, heparin, low molecular weightheparin, hirugen, hirulos, hirudine, interferons, interleukins,cytokines, mono and polyclonal antibodies, chemotherapeutic agents,vaccines, glycoproteins, bacterial toxoids, growth hormones, parathyroidhormone (PTH), calcitonins, insulin like growth factors (IGF), glucagonlike peptides (GLP-1 and GLP-2), steroids and retinoids, injectablelarge molecule antibiotics, protein based thrombolytic compounds,platelet inhibitors, DNA, gene therapeutics, RNA and antisenseoligonucleotides.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

When developing new pharmaceutical formulations, it is desirable toprovide dosage forms suitable for administering proteinic and peptidicdrugs to humans and animals through oral, nasal, pulmonary andtransdermal mucosal routes and to allow easy accessibility to the sitesof administration. Local absorption of macromolecular drugs is desirableover a prolonged period to maximize drug absorption. Furthermore, it isdesirable to minimize tissue damage and provide acceptable tissuecompatibility of the dosage form. It is preferable to provide systemswhich are pain free and easy to be administered with great flexibility,in order to gain high acceptance and compliance of any therapy bypatients.

It has been found that macromolecular drugs may be administered in mixedliposomal formulations in which particle sizes (1 to 4 nm) are smallerthan any pores of mucosal surfaces.

The present invention provides an improved method for delivery ofmacromolecular (high molecular weight) pharmaceutical agents,particularly through the skin or membranes in the nose, mouth, lungs,vagina or rectum. The preferred delivery is through oral and nasalcavities. The pharmaceutical agents cover a wide spectrum of agents,including proteins, peptides, hormones, vaccines and drugs. Themolecular weights of the macromolecular pharmaceutical agents arepreferably above 1000, especially between 1000 and 2 000 000.

For example, hormones which may be administered with the presentinvention include human growth hormones, parathyroid hormones,follicular stimulating hormones, luteinizing hormones, androgens,estrogens, prostoglandins, somatropins, gonadotropins, erythropoetin,interferons, interleukins, steroids and cytokines.

Vaccines which may be administered with the present invention includebacterial and viral vaccines such as vaccines for hepatitis A, hepatitisB, hepatitis C, influenza, tuberculosis, canary pox, chicken pox,measles, mumps, rubella, pneumonia, BCG, HIV, helicobector pylori andAIDS.

Bacterial toxoids which may be administered using the present inventioninclude diphtheria, tetanus, pseudonomas and mycobactrium tuberculosis.

Examples of specific cardiovascular or thromobolytic agents includeheparin, low molecular weight heparin, hirugen, hirulos and hirudine.

As will be understood, the concentration of the pharmaceutical agent isan amount sufficient to be effective in treating or preventing adisorder or to regulate a physiological condition in an animal or human.The concentration or amount of pharmaceutical agent administered willdepend on the parameters determined for the agent and the method ofadministration, e.g. oral, nasal, transdermal, pulmonary.

Preferred methods of forming mixed non-phospholipid membrane mimeticamphiphiles and phospholipid are based on the phase behaviour of lipidamphiphiles and phospholipids. Such methods use high turbulence or highshear methods of mixing, e.g. turbines or high velocity nozzles. Forexample, the membrane-mimetic amphiphiles may be injected at highvelocity, e.g. through nozzles, into an aqueous phase of thephospholipid. Alternatively, the membrane mimetic amphiphiles and thephospholipids may be mixed in a mixing chamber into which thephospholipids are injected at high velocity through one or more nozzlesand the membrane-mimetic amphiphiles are also injected at high velocitythrough one or more nozzles. Other ingredients, such as sodium laurylsulphate, protease inhibitors may be premixed with either themembrane-mimetic amphiphile or the phospholipid. The velocity and mixingof the two liquids depends in part on the viscosities of the materialsand nozzle diameters, e.g. 10 to 15 m/s through 0.5 to 1.0 mm diameternozzle apertures. Typically the ratio of the membrane-mimetic amphiphileaqueous solution to the phospholipid solution is about 5:1 to about 20:1and the temperature of mixing is typically from about 10° C. to 20° C.

It may sometimes be necessary to heat the membrane-mimetic amphiphilesand other ingredients in order to yield a homogeneous aqueous solutionprior to mixing with the phospholipids. The nature of the proteinicpharmaceutical may also dictate the temperature range at which mixingmay take place. The temperature of mixing is typically room temperatureor below, but may be higher than room temperature for certainformulations. The resulting formulation contains multi-lamellarliposomal vesicles. If the formulation has been heated during mixing, itis sometimes desirable to cool the mixture while still being mixed, inorder to assist in the formation of the multi-lamellar vesicles.

Mixed multi-lamellar vesicles formed by the present process are verysmall in size, e.g. less than 10 nm, and are stable under most storageconditions.

Preferably, the membrane-mimetic amphiphile solution is injected intothe phospholipid solution through tangentially placed nozzles in a smallcylindrical mixing chamber. Preferably, one or two nozzles are used forthe membrane-mimetic amphiphile solution and one or two alternatingnozzles for the phospholipid solution. The two liquids are preferablydelivered to the nozzles by flow-controlled positive displacement pumps.

Although the present invention has such wide applicability, theinvention is described hereinafter with particular reference to insulinand its analogues, which are used for the treatment of diabetes.

In the case of insulin, which is intended for administration throughnasal or oral cavities, an aqueous buffer solution may be made first byadding aqueous alkali metal lauryl sulphate to powdered insulin, andthen stirring until the powder is dissolved and a clear solution isobtained. The buffer solution may also contain sodium salicylate.Typical concentrations of sodium salicylate and sodium lauryl sulphatein the aqueous solution are about 3 to 20 wt./wt. % of each compound inthe solution. Typically, insulin is present in the solution in an amountwhich will give a concentration of about 2 to 4 wt./wt. % of the finalformulation.

The buffer solution is then added to liquid which comprises amembrane-mimetic amphiphile or a phospholipid while mixing vigorously,to form multi-lamellar liposomal vesicles.

The membrane-mimetic amphiphile is selected from the group consisting ofhyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,lauramidopropyl betain, lauramide monoisopropanolamide, sodiumcocoamphopropionate, bishydroxypropyl dihydroxypropyl stearammoniumchloride, polyoxyethylene dihydroxypropyl stearammonium chloride,dioctadecyldimethylammonium chloride, sulphosuccinates, stearamide DEA,gamma-linoleic acid, borage oil, evening of primrose oil, monoolein,sodium tauro dihydro fusidate, fusidic acid, alkali metal isostearyllactylates, alkaline earth metal isostearyl lactylates, panthenyltriacetate, cocamidopropyl phosphatidyl PG-diammonium chloride,stearamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropylphosphatidyl PG-diammonium chloride, borage amidopropylphosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid,trihydroxy-oxo-cholanylglycine and alkali metal salts thereof,octylphenoxypolythoxyethanol, polydecanol X-lauryl ether, polydecanolX-oleyl ether, wherein X is from 9 to 20, and combinations thereof.

The phospholipid is selected from the group consisting of phospholipidGLA, phosphatidyl serine, phosphatidylethanolamine,inositolphosphatides, dioleoylphosphatidylethanolamine, sphingomyelin,ceramides, cephalin, triolein, unsaturated lecithin, saturated lecithinand lysolecithin.

Each of the membrane-mimetic amphiphiles and phospholipids are presentin a concentration of from 1 to 10 wt./wt. % of the total formulation.

Preferred salts of hyaluronic acid are alkali metal hyaluronates,alkaline earth hyaluronates and aluminium hyaluronate. The preferredsalt is sodium hyaluronate. The preferred concentration of hyaluronicacid or pharmaceutically acceptable salts of hyaluronic acid is from 1to 5 wt./wt. % of the total formulation. An even more preferred range isfrom 1.5 to 3.5 wt./wt. % of the total formulation.

Other ingredients may be added to the liposomal solution. For example,flavouring agents, antioxidants, salts, protease inhibitors or otherpharmaceutically acceptable compounds may be added.

In general the size of the multi-lamellar liposomal vesicle particles isabout from 1 to 10 nm, and preferably from 1 to 5 nm. Such a sizedistribution ensures effective absorption of the formulation, andtherefore the pharmaceutical agent, through the membranes, for examplethe membranes in the oral and nasal cavities.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the nasal and oral cavities, it is often desirable to increase,e.g. double or triple, the dosage which is normally required throughinjection of administration through the gastrointestinal tract.

As will be understood, the amount of each component of the formulationwill vary depending on the pharmaceutical agent and the site ofapplication.

For oral application, sodium lauryl sulphate and sodium edetate areinsufficient on their own and must be combined with at least onemembrane-mimetic amphiphile and at least one phospholipid to promote theoral absorption of macromolecules to achieve therapeutic effects.

The oral formulations may be mixed with a suitable propellant anddelivered with a suitable applicator.

Preferred formulations oral or nasal application have the followingcombinations, in addition to sodium lauryl sulphate:

i) sodium salt of trihydroxy-oxo-cholanyl glycine, sphingomyelin andstearamide DEA;

ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA;

iii) phospholipid GLA, polydecanol 9-lauryl ether andoctylphenoxyethoxyethanol;

iv) ceramide and stearamidopropyl phosphatidyl PG-diammonium chloride;

v) borage amidopropyl phosphatidyl PG-diammonium chloride and lecithin;

vi) octylphenoxypolyethoxyethanol and saturated lecithin;

vii) lecithin, evening of primrose oil andtrihydroxy-oxo-cholanylglycine;

viii) sodium hyaluronate, trihydroxy oxo-cholanylglycine, lecithin andevening of primrose oil; and

ix) saturated lecithin, sodium hyaluronate, and evening of primrose oil.

Some preferred compositions for transdermal application have thefollowing absorption enhancing compound combinations, in addition tosodium lauryl sulphate and sodium edetate: i) sodium hyaluronate,saturated lecithin, glycolic acid and propylene glycol; ii) sodiumhyaluronate, sphingomyelin, glycolic acid and propylene glycol.

For topical applications, enhanced skin penetration can be obtained witha combination of glycolic lactic acid propylene glycol with theliposomes.

The therapeutic compositions of the present invention can be stored atroom temperature or at cold temperature. Storage of proteinic drugs ispreferable at a cold temperature, e.g. 4° C., to prevent degradation ofthe drugs and to extend their shelf life.

As indicated hereinbefore, generally, oral, pulmonary, transdermal andnasal are the favoured sites of the administration but the compositioncan be applied to the rectal and vaginal mucosa. According to thephysiologically active peptide or protein used, the dosage form and thesite of administration a specific administration method can be selected.

The composition of this invention is generally prepared as microfinemulti-lamellar liposomal vesicle particles (1 to 10 nm or less) by thevirtue of its preparation methods used and combinations suitablecharacteristics of the membrane mimetic amphiphiles and phospholipids.

Administration of the formulation is by methods generally known in theart. For oral and nasal application, sprays are preferable. Othermethods include the use of drops, chewable tablets, chewable gum,suppositories, lotions and ointments. Utilization of atomizer or aerosolspray devices (metered dose inhalers or nebulizers) can be used tofurther reduce the particle size for effective inhalation from the nasalor oral cavity so the drug may successfully reach to the specific site,especially the lungs, and be absorbed.

It is also possible to utilize a drug delivery system such that anenteric coating is applied to the gelatin capsule to cause the lipsomesto be released only in the duodenum or in the proximity of the largeintestine and not in the stomach.

The invention is illustrated by reference to the following examples.

EXAMPLE 1

26 000 units (1000 mg) of insulin crystals were suspended in 150 mL 0.3Mhydrochloric acid and the solution was stirred to dissolve the crystalscompletely. The pH was adjusted to 7.0 by neutralizing with 0.3M sodiumhydroxide. The final volume was adjusted to 260 mL to give 100 units/mLinsulin concentration

To 10 mL of insulin solution, 50 mg of sodium lauryl sulphate was addedand dissolved completely. In 50 mL of water, 50 mgtrihydroxy-oxo-cholanylglycine and 50 mg polydecanol 20-oleyl ether wereadded and dissolved and then mixed with the insulin solution. Thismixture was then sprayed under pressure into a 1 wt. % solution ofphospholipid GLA to form mixed lipsomes. This procedure gave a mixedamphiphile insulin solution with 50 units/mL.

The structure of the mixed amphiphile insulin was examined under a lightmicroscope and the particle size was analyzed by laser light scattering.The average particle size was estimated to be about 2 to 10 nm.

In one set of tests, ten diabetic human volunteers who normally tookinsulin by injection three times a day, were studied. The volunteerswere tested with insulin, taken orally. The volunteers fasted frommidnight prior to the test, with no food being taken during the 4 hourstudy.

Each of the volunteers received 10 units insulin. In one test, the oralinsulin was administered with a metered dose spray. In another test, theinsulin was administered by injection. Blood glucose levels, in mmol/L,were monitored every 15-30 minutes by Bayer's Glucometer Elite.

The average results for the ten volunteers, of the trial were asfollows:

TABLE I Time Oral Insulin Injection (minutes) (10 units) (10 units) 011.0 10.5 15 10.6 10.5 30 10.2 10.4 45 9.3 10.2 60 8.6 9.5 90 7.0 8.2120 6.5 6.8 150 5.9 5.5 180 5.1 4.7

The results show that the oral insulin formulation, within the scope ofthe present invention, at an equivalent dosage, is comparable with theinjected insulin.

EXAMPLE II

To 10 mL of the insulin solution prepared in Example I, 50 mg of sodiumlauryl sulphate was added and dissolved completely. In 50 mL of water,50 mg lauramidopropyl betain and 50 mg polydecanol 9-lauryl ether wereadded and dissolved and then mixed with the insulin solution. Thismixture was then sprayed under pressure into a 1 wt. % solution ofPhospholipon-H (trade mark) saturated lecithin, to form mixed lipsomes.This procedure gave a multilamellar, mixed amphiphile insulin solutionwith 50 units/mL.

The structure of the multilamellar, mixed amphiphile insulin wasexamined under a light microscope and the particle size was analyzed bylaser light scattering. The average particle size was estimated to beabout 2 to 10 nm.

In one set of tests, ten healthy human volunteers were studied. Thevolunteers were tested with insulin, taken orally and taken byinjection. The volunteers fasted from midnight prior to the test, withno food being taken during the 4 hour study.

Each of the volunteers received 10 units insulin. In one test, the oralinsulin was administered with a metered dose spray. In another test, theinsulin was administered by injection. Blood glucose levels, in mmol/L,were monitored every 30 minutes by Bayer's Glucometer Elite.

The average results for the ten volunteers, of the trial were asfollows:

TABLE II Time Oral Insulin Injection (minutes) (10 units) (10 units) 05.5 5.3 30 5.0 5.2 60 4.6 4.2 90 4.2 3.8 120 4.0 3.6 150 3.6 3.3 180 3.13.0

The results show that the oral insulin formulation, within the scope ofthe present invention, at an equivalent dosage, is comparable with theinjected insulin.

EXAMPLE III

To 10 mL of the insulin solution prepared in Example I, 50 mg of sodiumlauryl sulphate was added and dissolved completely. This mixture wasthen sprayed under pressure into a 1 wt. % solution of Phospholipon-H(trade mark) saturated lecithin to form mixed lipsomes. This proceduregave a multilamellar, mixed amphiphile insulin solution with 50units/mL.

This composition, which is outside the scope of the present invention,was tested on 10 healthy volunteers and compared to injected insulin, asin Example II.

The average results for the ten volunteers, of the trial were asfollows:

TABLE III Time Oral Insulin Injection (minutes) (10 units) (10 units) 05.7 5.9 30 5.8 5.7 60 5.5 5.0 90 5.4 4.8 120 5.3 4.3 150 5.4 3.8 180 5.33.2

The results show that the oral insulin formulation, outside the scope ofthe present invention, at an equivalent dosage, had little effect. Thisis probably because the insulin was not absorbed, and degraded faster.

EXAMPLE IV

To 10 mL of the insulin solution prepared in Example I, 100 mg of sodiumlauryl sulphate was added and dissolved completely.

This composition, which is outside the scope of the present invention,was tested on 10 healthy volunteers and compared to injected insulin, asin Example II.

The average results for the ten volunteers, of the trial were asfollows:

TABLE IV Time Oral Insulin Injection (minutes) (10 units) (10 units) 06.1 5.9 30 6.0 5.7 60 5.8 5.2 90 5.7 4.7 120 5.6 4.3 150 5.5 3.7

The results show that the oral insulin formulation, outside the scope ofthe present invention, at an equivalent dosage, had little effect.

EXAMPLE V

10 mL of the insulin solution prepared in Example I was added to a 1 wt.% solution of Phospholipon-H saturated lecithin.

This composition, which is outside the scope of the present invention,was tested on 10 healthy volunteers and compared to injected insulin, asin Example II.

The average results for the ten volunteers, of the trial were asfollows:

TABLE V Time Oral Insulin Injection (minutes) (10 units) (10 units) 06.2 5.9 30 6.3 5.6 60 6.2 5.0 90 6.4 4.6 120 6.5 4.1 150 6.4 3.8 180 6.53.2

The results show that the oral insulin formulation, outside the scope ofthe present invention, at an equivalent dosage, had no effect.

EXAMPLE VI

To 10 mL of the insulin solution prepared in Example I, 50 mg of sodiumlauryl sulphate was added and dissolved completely. In 50 mL of water,50 mg trihydroxy-oxo-cholanylglycine and 50 mg stearamide DEA were addedand dissolved and then mixed with the insulin solution. This mixture wasthen sprayed under pressure into a 1 wt. % solution of sphingomyelin, toform mixed micelles. This procedure gave a mixed amphiphile insulinsolution with 50 units/mL.

The structure of the mixed amphiphile insulin was examined under a lightmicroscope and the particle size was analyzed by laser light scattering.

This composition, which is within the scope of the present invention,was tested on 10 diabetic volunteers and compared to injected insulin,as in Example I.

The average results for the ten volunteers, of the trial were asfollows:

TABLE VI Time Oral Insulin Injection (minutes) (10 units) (10 units) 07.8 8.0 30 6.5 7.0 60 5.3 6.0 90 5.1 5.0 120 4.8 4.6 150 4.1 4.2 180 3.63.5

The results show that the oral insulin formulation, within the scope ofthe present invention, at an equivalent dosage, is comparable with theinjected insulin.

EXAMPLE VII

To 10 mL of the insulin solution prepared in Example I, 100 mg of sodiumlauryl sulphate was added and dissolved completely. In 50 mL of water,100 mg sodium hyaluronate, 0.5 mL glycolic acid and 0.5 mL propyleneglycol were added and dissolved and then mixed with the insulinsolution. This mixture was then sprayed under pressure into a 1 wt. %solution of Phospholipon-H (trade mark) saturated lecithin, to formmixed lipsomes.

In one set of tests, ten healthy human volunteers were studied. Thevolunteers were tested with insulin, applied topically and taken byinjection. The volunteers fasted from midnight prior to the test, withno food being taken during the 4 hour study.

Each of the volunteers received 10 units insulin. In one test, theinsulin was administered topically to a 2 cm² area of the back of thehand. In another test, the insulin was administered by injection. Bloodglucose levels, in mmol/L, were monitored every 30 minutes by Bayer'sGlucometer Elite.

The average results for the ten volunteers, of the trial were asfollows:

TABLE II Time Topical Insulin Injection (minutes) (10 units) (10 units)0 5.5 5.3 30 5.3 5.3 60 5.0 5.0 90 4.9 4.6 120 4.8 4.3 150 4.7 4.0 1804.5 3.8

The results show that the topical insulin formulation, within the scopeof the present invention, at an equivalent dosage, is comparable withthe injected insulin.

What is claimed is:
 1. A mixed liposome pharmaceutical formulation withmultilamellar vesicles, comprising a proteinic pharmaceutical agent,water, an alkali metal lauryl sulphate in a concentration of from 1 to10 wt./wt. % of the total formulation, at least one membrane-mimeticamphiphile and at least one phospholipid, wherein the membrane-mimeticamphiphile is selected from the group consisting of hyaluronic acid,pharmaceutically acceptable salts of hyaluronic acid, lauramidopropylbetain, lauramide monoisopropanolamide, sodium cocoamphopropionate,bishydroxypropyl dihydroxypropyl stearammonium chloride, polyoxyethylenedihydroxypropyl stearammonium chloride, dioctadecyldimethylammoniumchloride, sulphosuccinates, stearamide DEA, gamma-linoleic acid, borageoil, evening of primrose oil, monoolein, sodium tauro dihydro fusidate,fusidic acid, alkali metal isostearyl lactylates, alkaline earth metalisostearyl lactylates, panthenyl triacetate, cocamidopropyl phosphatidylPG-diammonium chloride, stearamidopropyl phosphatidyl PG-diammoniumchloride, borage amidopropyl phosphatidyl PG-diammonium chloride, borageamidopropyl phosphatidylcholine, polysiloxy pyrrolidone linoleylphospholipid, trihydroxy-oxo-cholanylglycine and alkali metal saltsthereof, and octylphenoxypolythoxyethanol, polydecanol X-lauryl ether,polydecanol X-oleyl ether, wherein X is from 9 to 20, and combinationsthereof, and wherein the phospholipid is selected from the groupconsisting of phospholipid GLA, phosphatidyl serine,phosphatidylethanolamine, inositolphosphatides,dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin,triolein, lecithin, saturated lecithin and lysolecithin, andcombinations thereof, and wherein each membrane mimetic amphiphile andphospholipid is present in a concentration of from 1 to 10 wt./wt. % ofthe total formulation, and the total concentration of membrane mimeticamphiphiles and phospholipids is less than 50 wt./wt. % of theformulation.
 2. A formulation according to claim 1 wherein the alkalimetal lauryl sulphate is sodium lauryl sulphate.
 3. A formulationaccording to claim 1 wherein there are at least two membrane mimeticamphiphiles.
 4. A formulation according to claim 1 wherein themembrane-mimetic amphiphile is selected from the group consisting ofhyaluronic acid, pharmaceutically acceptable salts of hyaluronic acidand mixtures thereof, the concentration such absorption enhancingcompound being from about 1 to about 5 wt./wt. %.
 5. A formulationaccording to claim 1 which contains sodium lauryl sulphate andcombinations selected from the group consisting of: i) sodium salt oftrihydroxy-oxo-cholanyl glycine, sphingomyelin and stearamide DEA; ii)sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipid GLA;iii) phospholipid GLA, polydecanol 9-lauryl ether andoctylphenoxyethoxyethanol; iv) ceramide and stearamidopropylphosphatidyl PG-diammonium chloride; v) borage amidopropyl phosphatidylPG-diammonium chloride and lecithin; vi) octylphenoxypolyethoxyethanoland saturated lecithin; and vii) lecithin, evening of primrose oil andtrihydroxy-oxo-cholanylglycine; viii) sodium hyaluronate, trihydroxyoxo-cholanylglycine, lecithin and evening of primrose oil; ix) sodiumhyaluronate, saturated lecithin, and evening of primrose oil; x) sodiumhyaluronate and saturated lecithin; and xi) sodium hyaluronate andsphingomyelin.
 6. A formulation according to claim 1 wherein thepharmaceutical agent is selected from the group consisting of insulin,heparin, low molecular weight heparin, hirugen, hirulos, hirudine,interferons, interleukins, cytokines, mono and polyclonal antibodies,chemotherapeutic agents, vaccines, glycoproteins, hormones, bacterialtoxoids, growth hormones, calcitonins, insulin like growth factors(IGF), glucagon like peptides (GLP-1 or GLP-2), steroids and retinoids,injectable large molecule antibiotics, protein based thrombolytlccompounds, platelet inhibitors, DNA, Gene therapeutics, RNA andantisense oligonucleotides.
 7. A process for making a pharmaceuticalcomposition comprising: mixing in a high shear mixer a proteinicpharmaceutical agent, water, an alkali metal lauryl sulphate in aconcentration of from 1 to 10 wt./wt. % of the total formulation, atleast one membrane-mimetic amphiphile and at least one phospholipid,wherein the membrane-mimetic amphiphile is selected from the groupconsisting of hyaluronic acid, pharmaceutically acceptable salts ofhyaluronic acid, lauramidopropyl betain, lauramide monoisopropanolamide,sodium cocoamphopropionate, bishydroxypropyl dihydroxypropylstearammonium chloride, polyoxyethylene dihydroxypropyl stearammoniumchloride, dioctadecyldimethylammonium chloride, sulphosuccinates,stearamide DEA, gamma-linoleic acid, borage oil, evening of primroseoil, monoolein, sodium tauro dihydro fusidate, fusidic acid, alkalimetal isostearyl lactylates, alkaline earth metal isostearyl lactylates,panthenyl triacetate, cocamidopropyl phosphatidyl PG-diammoniumchloride, stearamidopropyl phosphatidyl PG-diammonium chloride, borageamidopropyl phosphatidyl PG-diammonium chloride, borage amidopropylphosphatidylcholine, polysiloxy pyrrolidone linoleyl phospholipid,trihydroxy-oxo-cholanylglycine and alkali metal salts thereof, andoctylphenoxypolythoxyethanol, polydecanol X-lauryl ether and polydecanolX-oleyl ether, wherein X is from 9 to 20, and wherein the phospholipidis selected from the group consisting of phospholipid GLA, phosphatidylserine, phosphatidylethanolamine, inositolphosphatides,dioleoylphosphatidylethanolamine, sphingomyelin, ceramides, cephalin,triolein, lecithin, saturated lecithin and lysolecithin, and whereineach membrane mimetic amphiphile and phospholipid is present in aconcentration of from 1 to 10 wt./wt. % of the total formulation, andthe total concentration of membrane mimetic amphiphiles andphospholipids is less than 50 wt./wt. % of the formulation; said mixingbeing continued until the composition is in multilamellar vesicle form.8. A process according to claim 7 wherein the membrane-mimeticamphiphile is selected from the group consisting of hyaluronic acid,pharmaceutically acceptable salts of hyaluronic acid and mixturesthereof, the concentration such absorption enhancing compound being fromabout 1 to about 5 wt./wt. %.
 9. A process according to claim 7 whereinthe alkali metal lauryl sulphate is sodium lauryl sulphate.
 10. Aprocess according to claim 7 wherein phospholipids and amphiphilescomprise a combination selected from the group consisting of: i) sodiumsalt of trihydroxy-oxo-cholanyl glycine, sphingomyelin and stearamideDEA; ii) sodium salt of trihydroxy-oxo-cholanyl glycine and phospholipidGLA; iii) phospholipid GLA, polydecanol 9-lauryl ether andoctylphenoxyethoxyethanol; iv) ceramide and stearamidopropylphosphatidyl PG-diammonium chloride; v) borage amidopropyl phosphatidylPG-diammonium chloride and lecithin; vi) octylphenoxypolyethoxyethanoland saturated lecithin; and vii) lecithin, evening of primrose oil andtrihydroxy-oxo-cholanylglycine; viii) sodium hyaluronate, trihydroxyoxo-cholanylglycine, lecithin and evening of primrose oil; ix) saturatedlecithin, sodium hyaluronate, and evening of primrose oil; x) saturatedlecithin and sodium hyaluronate; and xi) sodium hyaluronate andsphingomyelin.
 11. A process according to claim 7 wherein the proteinicpharmaceutical agent is selected from the group consisting of insulin,heparin, low molecular weight heparin, hirugen, hirulos, hirudine,interferons, interleukins, cytokines, mono and polyclonal antibodies,chemotherapeutic agents, vaccines, glycoproteins, bacterial toxoids,hormones, calcitonins, insulin like growth factors (IGF), glucagon likepeptides (GLP-1 or GLP-2), steroids, retinoids, large moleculeinjectable antibiotics, protein based thrombolytic compounds, plateletinhibitors, DNA, RNA, gene therapeutics and antisense oligonucleotides.12. A process according to claim 7 wherein the method of mixing is ahigh turbulence or high shear method of mixing.
 13. A process accordingto claim 12 selected from the group consisting of i) injecting thephospholipid, in liquid form, at high velocity through at least onenozzle into an aqueous phase of the membrane-mimetic amphiphile, ii)injecting the membrane-mimetic amphiphile, in liquid form, at highvelocity through at least one nozzle into an aqueous phase of thephospholipid, and iii) injecting the phospholipid, in liquid form, athigh velocity through at least one nozzle and the membrane mimeticamphiphile, in liquid form, at high velocity through at least one nozzleinto a mixing chamber; and wherein the alkali metal lauryl sulphate ispresent with either the phospholipid or membrane-mimetic amphiphile. 14.A process according to claim 13 wherein the velocity of the phospholipidand amphiphile liquids is from 0 to 15 m/s and the nozzle apertures arefrom 0.5 to 1.0 mm in diameter.
 15. A process according to claim 12wherein the ratio of the membrane-mimetic amphiphile aqueous solution tothe phospholipid solution is about 5:1 to about 20:1.
 16. A processaccording to claim 13 wherein the ratio of the membrane-mimeticamphiphile aqueous solution to the phospholipid solution is about 5:1 toabout 20:1.